Toll-like Receptor3-mediated Induction of Chemokines in Salivary Epithelial Cells

Jingchao Li; Mi Young Jeong; Ji Hyun Bae; Yong Hwan Shin; Meihong Jin; Sung Min Hang; Jeong Chai Lee; Sung Joong Lee; Kyungpyo Park

doi:10.4196/kjpp.2010.14.4.235

Journal List > Korean J Physiol Pharmacol > v.14(4) > 1025684

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Li, Jeong, Bae, Shin, Jin, Hang, Lee, Lee, and Park: Toll-like Receptor3-mediated Induction of Chemokines in Salivary Epithelial Cells

Original Article

Korean J Physiol Pharmacol 2010;14(4):235-240.

Published online: 18 February 2010

DOI: https://doi.org/10.4196/kjpp.2010.14.4.235

Toll-like Receptor3-mediated Induction of Chemokines in Salivary Epithelial Cells

Jingchao Li^1,^∗, Mi Young Jeong^1,^∗, Ji Hyun Bae¹, Yong Hwan Shin¹, Meihong Jin¹, Sung Min Hang¹, Jeong Chai Lee², Sung Joong Lee¹, Kyungpyo Park^1,

¹Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Korea

²R&D Center of Medi-Future, Seongnam 462-721, Korea

Corresponding to: Kyungpyo Park, Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, 28, Yeongeon-dong, Jongno-gu, Seoul 110-749, Korea. (Tel) 82-2-740-8658, (Fax) 82-2-762-5107, (E-mail) kppark@snu.ac.kr

^∗ These authors equally contribute to this work.

Received 2 August 2010 Revised 10 August 2010 Accepted 11 August 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Toll-like receptors (TLRs) functionally expressed in salivary epithelial cells, but their roles remain elusive. Among TLRs family, TLR3 is activated by dsRNA, a byproduct of viral infection. The aim of this study was to investigate the role of TLR3 in the inflammatory immune responses using HSG cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and ELISA were performed to identify expression of TLRs and TLR3-mediated chemokine inductions. The chemotaxis assay of activated T lymphocytes was also performed. Treatment of HSG cells with polyinosinic: polycytidylic acid (poly(I:C)) significantly increased interferon-γ-inducible protein 10 (IP-10), interferon-inducible T-cell α chemoattractant (I-TAC), and regulated on activation, normal T-cells expressed and secreted (RANTES) gene expressions in a concentration-dependent manner. Anti-TLR3 antibody blocked the increases of IP-10 and I-TAC genes. Poly(I:C)-induced increases of IP-10 and I-TAC were also confirmed at protein levels from cell lysates, but their release into extracellular medium was detected only in IP-10. We found that the culture media from HSG cells stimulated with poly(I:C) significantly increases T lymphocyte migration. Our results suggest that TLR3 plays an important role in chemokine induction, particularly IP-10, in salivary epithelial cells.

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Keywords: Toll-like receptors, HSG cells, Chemokine, Poly(I:C), IP-10

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Fig. 1.

mRNA expression of Toll like receptor (TLR) subtypes in human submandibular glands (hSMG) and HSG cell lines. (A) Strong mRNA expression of TLR1, 3, 5, and 9 and weak expression of TLR2, 4, and 6 in hSMG. M; marker protein, Pwon600DNA/ECOR1+HinfI Digest N; negative control. (B) Strong mRNA expression of TLR1, 3 and 6, and weak expressions TLR4 and 5 in HSG cell lines.

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Fig. 2.

Poly(I:C)-induced mRNA expressions of IP-10, I-TAC, and RANTES in HSG cells. The means±S.E.M of three independent experiments are shown. (A∼C) Increase in chemokine gene expression in a concentration-dependent manner. Treatment of cells with 10 μg/ml poly(I:C) significantly increased (p<0.01, indicated by ^∗) induction of IP-10, I-TAC, and RANTES by 26.5±1.2, 28.6±10.0, and 5.6±0.9 folds (dark grey bar in A, B, C), respectively, compared to the control (Con). The high concentration of poly(I:C), 40 μg/ml, further increased inductions of these chemokines by 209.3±19.7, 216.2±88.3, and 57.6±4.7 folds, respectively (black bar in A, B, C). (D∼F) Expression levels of chemokine genes in a various incubation time with 10 μg/ml of poly(I:C). Peak increases of all three chemokine expressions were observed after incubation of cells with poly(I:C) for 6 hrs by 68.3±2.3, 53±0.6, and 13.2±0.7 folds (p<0.001, indicated by ^∗∗), respectively (hatched bar in D, E, and F).

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Fig. 3.

Effects of TLR3 antibody on the IP-10 (A), I-TAC (B), and RANTES (C) mRNA expression induced by 10 μg/ml poly(I:C) for 6 hrs. The means±S.E.M of three independent experiments are shown. Poly(I:C)-induced inductions of IP-10, I-TAC and RANTES mRNA were decreased to 4.7±0.09, 1.4±0.3, and 14±5 folds, respectively, by the addition of 20 μg/ml TLR3 antibody (hatched bars). TLR3 antibody significantly decreased IP-10 and I-TAC mRNA expression (p<0.001, indicated by ^∗∗), but not RANTES. Addition of mouse IgG (black bars) instead of TLR3 antibody has no effect on the expression of the three chemokine genes.

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Fig. 4.

Poly(I:C)-induced chemokine release and migration of activated T lymphocytes. The means±S.E.M of three independent experiments are shown. (A) I-TAC protein concentration from cell lysates in the control (C, 215±9.2 pg/ml), PMB (217.7±16.7 pg/ml), and poly(I:C) treatment groups. 10 μg/ml of poly(I:C) significantly increased protein concentration to 312.6±26.4 pg/ml (p<0.01, indicated by ^∗), compared to the control. (B) I-TAC protein concentrations in culture medium in control (C), PMB, and poly(I:C) treatment groups with 10, 20, and 40 μg/ml concentrations. The amount of I-TAC protein in the poly(I:C) treatment group was not significantly different, compared to the control or PMB groups (p> 0.1). Note the different scale of Y-axis. (C) IP-10 protein concentrations in control (313.1±20.1 pg/ml), PMB (262.6±13.1 pg/ml), and poly(I:C) treatment groups in culture media. 10 μg/ml of poly(I:C) significantly increased protein concentration to 837.4±58.7 pg/ml (p< 0.001, indicated by ^∗). (D) % migration of activated T lymphocytes induced by three different incubation media: untreated (control, white bar, 17.6±1.5%, n=3), treated with 10 μg/ml poly(I:C) (grey bar, 31.3±2.0%, n=3), or plus antibodies against IP-10 (black bar, 18.3±1.5%, n=3). 10 μg/ml poly(I:C) significantly increased migration of activated T lymphocytes compared to the control (p<0.01, indicated by ^∗), and addition of anti-IP-10 Ab completely blocked the poly(I:C)-induced T lymphocyte migration.

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